Optimisation of mesenchymal stromal cells karyotyping analysis: implications for clinical use.

PURPOSE: The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use. BACKGROUND: MSC are being increasingly used in clinical trials for a number of diseases. Biosafety demonstration in all cases is mandatory. Unfortunately, current standard karyotyping methods fail to obtain enough number of evaluable metaphases. METHODS AND MATERIALS: In the present work, to optimise the yield of metaphases in MSC expanded in vitro, we have tested several conditions by modifying colcemid concentration (we have tested 0.01, 0.05 and 0.1 µg mL(-1) ) and exposure time (during 5, 15 and 24 h). We further applied these optimised conditions to 61 MSC expansions in GMP conditions for clinical use. RESULTS: Our results show that the highest number of metaphases was obtained when MSC were incubated with 0.05 µg mL(-1) of colcemid overnight (15 h), compared to the remaining experimental conditions. In most cases (59/61 cases) enough number of metaphases was obtained. And what is more relevant, only in one case a karyotypic abnormality was found (trisomy of chromosome 10), and cells were subsequently discarded for clinical use. CONCLUSION: We describe here an optimal method to obtain enough number of metaphases for karyotype analysis of in vitro expanded MSCs, what is essential for their clinical use in cell therapy programmes.

Effects of MSC coadministration and route of delivery on cord blood hematopoietic stem cell engraftment.

Hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) progenitors is increasingly being used. One of the problems that may arise after UCB transplantation is an impaired engraftment. Either intrabone (IB) injection of hematopoietic progenitors or mesenchymal stem cell (MSC) coadministration has been proposed among the strategies to improve engraftment. In the current study, we have assessed the effects of both approaches. Thus, NOD/SCID recipients were transplanted with human UCB CD34+ cells administered either intravenously (IV) or IB, receiving or not bone marrow (BM)-derived MSCs also IV or IB (in the right femur). Human HSC engraftment was measured 3 and 6 weeks after transplantation. Injected MSCs were tracked weekly by bioluminescence. Also, lodgment within the BM niche was assessed at the latter time point by immuno-fluorescence. Our study shows regarding HSC engraftment that the number of BM human CD45+ cells detected 3 weeks after transplantation was significantly higher in mice cotransplanted with human MSCs. Moreover, these mice had a higher myeloid (CD13+) engraftment and a faster B-cell (CD19+) chimerism. At the late time point evaluated (6 weeks), human engraftment was higher in the group in which both strategies were employed (IB injection of HSC and MSC coadministration). When assessing human MSC administration route, we were able to track MSCs only in the injected femurs, whereas they lost their signal in the contralateral bones. These human MSCs were mainly located around blood vessels in the subendosteal region. In summary, our study shows that MSC coadministration can enhance HSC engraftment in our xenogenic transplantation model, as well as IB administration of the CD34+ cells does. The combination of both strategies seems to be synergistic. Interestingly, MSCs were detected only where they were IB injected contributing to the vascular niche.

Caracterización de células madre de núcleo pulposo de disco intervertebral cervical y comparación con células de médula ósea de los mismos pacientes / Characterization of stem cells from nucleus pulposus of cervical intervertebral disc and compared with bone marrow cells from the same patients

Objetivo: Evaluar la presencia de células mesenquimales en el núcleo pulposo de disco intervertebral cervical y caracterizarlas comparativamente con las obtenidas de médula ósea de los mismos sujetos. Pacientes y metodología: Hemos realizado un estudio descriptivo con 14 pacientes que precisaron cirugía de artrodesis cervical. Se analizó la presencia de células mesenquimales (CSM) en el núcleo pulposo (NP) del disco, comparándolas cualitativamente con las de médula ósea (MO) de los mismos pacientes. Se aislaron y expandieron CSM, tanto de NP como de MO. Se realizaron los estudios de diferenciación multilineal in vitro de las células mesenquimales de ambas fuentes, hacia osteoblasto y adipocito, y caracterización inmunofenotípica por citometría de flujo. Resultados: Las células de ambos orígenes se diferencian in vitro hacia ambos tipos celulares, si bien la diferenciación adipocítica de las células procedentes del disco fue menor que las procedentes de MO. Tampoco se han demostrado diferencias en los marcadores inmunofenotípicos. Las células de ambas fuentes poseen los marcadores inmunofenotípicos característicos de las células mesenquimales. Conclusión: El NP de disco vertebral cervical degenerado contiene células troncales mesenquimales. Estas células son similares a las células de MO, con la excepción de su capacidad disminuida de diferenciación adipogénica (AU)

Objective: To evaluate the presence of mesenchymal cells in the nucleus pulposus (NP) of cervical discs and characterize them in comparison ot those obtained from the bone marrow of the same subjects. Patients and methods: We have performed a descriptive study with 14 patients requiring cervical fusion surgery. The presence of mesenchymal stem cells (MSCs) were analyzed in the NP from the disk and compared qualitatively with the bone marrow (BM) of the same patients. MSC were isolated and expanded for both NP and MO. We performed in vitro differentiation studies of mesenchymal cells from both sources, into osteogenic and adipogenic lines, and flow cytometric immunophenotyping. Results: We got differentiation towards both cell types, although adipocyte differentiation of disc-derived cells was decreased compared to those from BM. There were no differences in immunophenotypic markers. Cells from both sources have immunophenotypic markers characteristic of mesenchymal cells. Conclusion: The NP of degenerated cervical disc contains mesenchymal stem cells. These cells are quite similar to BM cells, with the exception of a diminished adipogenic differentiation capacity (AU)